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First published online 18 February 2004
doi: 10.1242/dev.01010

Development 131, 1197-1210 (2004)
Published by The Company of Biologists 2004
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Discrete gene sets depend on POU domain transcription factor Brn3b/Brn-3.2/POU4f2 for their expression in the mouse embryonic retina

Xiuqian Mu1, Phillip D. Beremand2, Sheng Zhao3, Rashmi Pershad4, Hongxia Sun1, Ann Scarpa1, Shuguang Liang1, Terry L. Thomas2 and William H. Klein1,*

1 Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
2 Department of Biology and The Laboratory for Functional Genomics, Texas A&M University, College Station, TX 77843-3285, USA
3 Department of Biomathematics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
4 Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA



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  		Fig. 1. Real-time PCR confirming the expression changes of subsets of genes in Brn3b-/- retina. The x-axis is relative fluorescence units (RFU), indicating PCR product accumulation, and the y-axis indicates PCR cycles. Wild type is red and Brn3b-/- is green. ß-actin serves as an RNA input control. Syntg4 is synaptotagmin 4. The accumulation of fluorescence for Brn3b in the Brn3b-/- sample near the end of the reaction is due to nonspecific amplification, as confirmed by the melting curve and agarose gel electrophoresis (data not shown). Fold changes obtained by real-time PCR are shown in Table 1.

 


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  		Fig. 2. In situ hybridization for subsets of genes reveals that these genes depend on Brn3b for expression in the retina through both cell-autonomous and non-cell-autonomous mechanisms. DIG-labeled probes for individual genes were hybridized to E14.5 Brn3b heterozygous (+/-) and null (-/-) sections across the eye. (A) Consistent with the microarray results, expression of the RGC-specific neurofilament 66 gene (Nf66) was not dependent on Brn3b. (B) Two examples, Dlx1 and cyclin D1 (Clnd1), whose expression did not coincide with Brn3b but was affected by the absence of Brn3b. In the heterozygous retina, both Dlx1 and cyclin D1 were expressed mostly in the progenitor cell layer. In the Brn3b-/- retina, Dlx1 was upregulated, most prominently in the RGCs, while cyclin D1 was downregulated. (C) Five examples RGC-specific genes downregulated in the absence of Brn3b: Gap43, synaptotagmin 13 (Syt13), neurofilament light-chain gene (Nfl), Hermes and myostatin/Gdf8.

 


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  		Fig. 3. Brn3b-dependent genes require Brn3b for expression only in the retina. Shown here are four genes (A, Rgcg1; B, synaptotagmin 4; C, persyn; D, Brn3a) whose expression was downregulated in the RGC layer in Brn3b-null (-/-) when compared with the heterozygous retina (+/-), but was not changed in other tissues. NC, nasal cavity; R, retina; DRG, dorsal root ganglia; SC, spinal cord.

 


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  		Fig. 4. The sonic hedgehog pathway is disrupted in Brn3b-/- retinas. (A) Real-time RT-PCR analysis of genes belonging to the sonic hedgehog pathway. Red is for wild-type and green is for Brn3b-/-. ß-actin was used as RNA input control. Three genes, sonic hedgehog (Shh), Gli1 and patched 2 (Ptch2), were significantly downregulated in Brn3b-/- retina, while other genes showed no change. (B) In situ hybridization of Shh, Gli1 and Smo on E14.5 Brn3b heterozygous (+/-) and null (-/-) sections. Gli1 and Smo were expressed in progenitor cells and Shh in RGCs. Gli1 and Shh were downregulated in Brn3b-/- retinas, while Smo showed no change.

 


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  		Fig. 5. Brn3b binds to a conserved element (SBRN3) in the Shh gene. (A) Alignment of a conserved region in the first intron of Shh in mouse, human, zebrafish and Fugu. Red letters indicate nucleotides not conserved among the species. Percentage of identity for each species when compared with the mouse sequence is indicated at the end of the sequences. The Brn3b-binding site (SBRN3) is underlined in the mouse sequence and is completely conserved in all species. The bottom shows the alignment of SBRN3 with the reported consensus Brn3b binding site. (B) Electrophoretic mobility shift assay using a GST-Brn3b POU fusion protein and a 32P-labeled oligonucleotide probe encompassing SBRN3. GST-3bPOU formed a specific complex (C), and formation of this complex was strongly inhibited by both an unlabeled Brn3b consensus oligonucleotide (100x) and the SBRN3 oligonucleotide (100x). A supershifted complex (S) was formed and retained in the loading well when anti-GST antibody was added. F is free SBRN3 probe.

 


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  		Fig. 6. SBRN3 mediates transcriptional activation by Brn3b in HEK 293 cells. On the left are schematics of the reporter constructs used in the transfection assay. A contains the minimal rat prolactin promoter (-36prl) alone (P, green box) upstream of the luciferase cDNA (Luc); B shows the conserved region (yellow box) in the first intron of mouse Shh sequence cloned upstream of -36prl; C is the same as B except that the SBRN3 site is mutated (indicated by a filled black circle); D contains three copies of the consensus Brn3-binding site (blue boxes) upstream of -36prl. On the right is the normalized luciferase activity (in arbitrary units) generated from the reporter constructs co-transfected into HEK 293 cells, with (+) or without (-) the Brn3b expression vector. Each bar represents the average of three replicate experiments. Fold change in luciferase activity for each construct in the presence of Brn3b is indicated at the top of the respective bars.

 


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  		Fig. 7. Alterations in Brn3b-dependent gene expression at different stages of retina development. Each column represents one developmental stage (E14.5, E16.5 and E18.5) and each horizontal line represents one cDNA clone. Fold changes (wild type/Brn3b-null) are color-coded according to the bar at the bottom of the figure; red represents downregulation in Brn3b-null retina and green represents upregulation. The cDNA clones were clustered according to similarities their expression patterns as shown by the tree of the right side of the figure. The four major clusters, A, B, C and D, are discussed in the text.

 

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© The Company of Biologists Ltd 2004